Most drug development targets catalyze phosphoryl-transfer to or from nucleotide triphosphates. Because catalysis changes their conformation, both affinity and selectivity for these targets depend on structural aspects that are changing rapidly precisely as they develop highest affinity. Thus, they are, necessarily, moving targets. Many such enzymes also transduce chemical free energy by linking hydrolysis of their purine triphosphate substrates to conformational changes used for cellular work and signaling. These enzymes include many that possess 1/2 folds described by Rossmann. Virtually all use a metal ion for catalysis. Our central hypothesis is that in enzymes whose conformational changes are responsible for free energy transduction the metal acts catalytically if, and only if, conformational changes reposition it. More formally, interactions of the Mg2+ ion from within the active site oppose catalysis, while longer-range interactions drive conformational motions from elsewhere in the protein, acting indirectly to change the Mg2+ coordination so that it can stabilize the chemical transition state. Preliminary work on B. stearothermophilus tryptophanyl-tRNA synthetase, TrpRS, shows conclusively that active-site protein-metal coupling opposes catalysis, in keeping with the hypothesis. To confirm the hypothesis, we seek positive evidence demonstrating synergistic interactions with the metal from a specific and highly conserved packing motif (the D1 Switch) common to all Rossmannoid enzymes (Aim 1). Thermodynamic cycles for several D1 point mutants, assayed with Mg2+ and Mn2+ have demonstrated significant synergistic coupling to the catalytic metal. A complete dataset may also support specific molecular mechanisms for this long-range coupling, thereby strengthening the hypothesis and broadening its impact on understanding molecular mechanisms of free- energy transduction. We discovered in preliminary work that Mn2+ also relaxes specificity of TrpRS for Trp vs. Tyr. In Aim 2, we will examine D1 (Aim 1) and D3 (specific to the Trp pocket) switch mutants to determine if this effect requires long-range coupling or arises only from properties of the metal. Insight into the mechanism of Mn2+-induced relaxation of specificity may have important implications for understanding the mutagenic affect of Mn2+ in polymerases. Finally, TrpRS also provides a superb model system to test whether or not incomplete factorial experimental design can reduce the total number of experiments necessary to parameterize predictive models for how allosteric protein functions change with combinatorial mutations (Aim 3). If we can draw valid, useful conclusions about the complex behavior of the D1 Switch from a small subset of the full factorial design of 127 genotypes, using similar innovative designs will enhance the experimental characterization of both related (a similar switch exists in CheY) and dissimilar phenomena. PUBLIC HEALTH RELEVANCE: A pervasive and unsolved problem in structural biology is how catalysis of purine triphosphate hydrolysis is coupled to conformational changes necessary for specificity, regulation, signaling, and biomechanics. Our work has raised a new possibility of an unexpected and potentially widespread coupling mechanism whereby Mg2+ can act catalytically if and only if the conformation changes. Testing this hypothesis by combinatorial mutagenesis a widely conserved conformational switching motif in Bacillus stearothermophilus Tryptophanyl-tRNA synthetase will likely establish new mechanistic paradigms linking transition-state stabilization by Mg2+ to domain movement via distributed use of ATP binding energy, with broad relevance to catalysis specificity, and free-energy transduction.